Interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), play an important role in the pathogenesis of many "age" and "inactivity"-related diseases, but the mechanism for age- and physical activity-induced differences in inflammatory cytokine production has not been elucidated. Toll-like receptor 4 (TLR4), in concert with CD14, transduces the lipopolysaccharide (LPS) signal, but no researchers have documented the effects of either aging or physical activity on TLR4. The broad objective of this application is to examine whether age or physical activity level influence the cell-surface expression of TLR4 and to determine to what extent these changes are related to differences in inflammatory cytokine production. The primary aims of the proposed research will be to assess the influence of age and physical activity level on TLR4 expression and to assess concomitant changes in LPS-stimulated inflammatory cytokine production, single-cell cytokine production and MAP kinase activation. Heat shock proteins (HSP60/72) are released from muscle following exercise and are known to bind and activate TLR4. Thus, we will also endeavor to determine the role heat shock proteins play in these relationships. It is hypothesized that both younger and physically active subjects will have both lower cell-surface expression of TLR4 and cytokine production than their older and more sedentary counterparts. We also hypothesize that LPS-stimulated MAP kinase activation will be proportional to cell-surface expression of TLR4, but will not be different between groups when alternative stimulation pathways are engaged (anti CD40). We will recruit 16 subjects into each of four groups: 18-35 years of age, physically inactive; 18-35, physically active; 65-85, physically inactive and 65-85, physically active. Blood samples will be obtained from subjects who have not exercised for 72 hours, following 24 h dietary control. Cell-surface expression of TLR4/CD14 and single-cell cytokine production (flow cytometry), in vitro cytokine stimulation (LPS, HSP72 and HSP60) and MAP kinase activation will be determined. A 2 x 2 factorial design with age (18-35 and 65-85) and physical activity level (active and inactive) as the two experimental factors, will be employed. These experiments will help to determine whether age and physical activity related differences in cytokine production are linked to TLR4 and whether heat shock proteins play a role in TLR4 regulation.